This invention relates to triphenylalkene derivatives and their use as selective estrogen receptor modulators (SERMs).
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice are incorporated by reference.
Estrogens have been known as female sex hormones. However, lately many tissue-specific properties for estrogens have been described in organs, which are not classically considered to be estrogen-sensitive or estrogen-responsive. During the menopause the secretion of estrogens is dramatically decreased. Subsequently elderly women develop commonly climacteric symptoms including hot flushes, sweating, insomnia, depression, headache, vaginal dryness, cardiovascular symptoms, urinary incontinence, swelling feeling, breast tenderness and fatigue. In long-term estrogen deficiency induces cardiovascular disorders and osteoporosis which increase the risk of bone fractures and hospitalizations, which are very expensive to the society. Estrogens are increasingly used for the treatment of climacteric symptoms, but on the other hand estrogen use increases the risk of uterine and breast cancers (Lobo, 1995). Estrogens are shown to be beneficial also in the prevention of Alzheimer""s disease (Henderson, 1997) and in the lowering of LDL-cholesterol values and thus preventing cardiovascular diseases (Grodstein and Stampfer, 1998). New therapies which would have the benefits of estrogens, but not the carcinogenic risks are requested. Selective estrogen receptor modulators (SERMs) have been developed to fulfill these requirements (Macgregor and Jordan, 1998). However, the presently used SERMs have properties which are far from optimal. E.g. raloxifen use is limited by its strong antiestrogenic properties, which cause and worsen the climacteric symptoms, although the effects on the bone are beneficial (Khovidhunkit and Shoback, 1999). It would be most desirable to develop tissue-specific estrogens, which could be used in women in the treatment of climacteric symptoms, osteoporosis, Alzheimer""s disease and/or cardiovascular diseases without the carcinogenic risk. At the best new SERMs could be given to men to protect against osteoporosis, cardiovascular diseases and Alzheimer""s disease without estrogenic adverse events (gynecomastia, decreased libido etc.).
One object of the present invention is to provide novel selective estrogen receptor modulators.
Another object of the present invention is to provide a pharmaceutical composition comprising an amount effective to produce a tissue specific estrogenic and/or antiestrogenic effect of said novel selective estrogen receptor modulator compound, or a stereoisomer, or a non-toxic pharmaceutically acceptable salt or ester thereof, and a pharmaceutically compatible acceptable carrier therefor.
An additional object of the present invention is to provide a method of producing a tissue specific estrogenic and/or antiestrogenic effect in a subject in which such an effect is desired which comprises administering to said subject said novel selective estrogen receptor modulator compound, or a stereoisomer, or a non toxic pharmaceutically acceptable salt or ester thereof in an amount sufficient to produce the desired effect.
Thus, according to one aspect this invention concerns novel selective estrogen receptor modulator compounds of the general formula: 
wherein R1 and R2, which are the same or different are
a) H, halogen, OCH3, OH; or 
where X is O, NH or S; and n is an integer from 1 to 4; and
R4 and R5, which are the same or different, are a 1 to 4 carbon alkyl, H, xe2x80x94CH2Cxe2x89xa1CH or xe2x80x94CH2CH2OH; or
R4 and R5 form an N-containing five- or six-membered ring or heteroaromatic ring; or
c) xe2x80x94Yxe2x80x94(CH2)nCH2xe2x80x94Oxe2x80x94R6
where Y is O, NH or S and n is an integer from 1 to 4; and
R6 is H, xe2x80x94CH2CH2OH, or xe2x80x94CH2CH2Cl; or
d) 2,3-dihydroxypropoxy, 2-methylsulfamylethoxy, 2-chloroethoxy, 1-ethyl-2-hydroxyethoxy, 2,2-diethyl-2-hydroxyethoxy or carboxymethoxy; and
R3 is H, halogen, OH or xe2x80x94OCH3;
stereoisomers thereof and non-toxic pharmaceutically acceptable salts and esters and mixtures thereof, provided that 
in the 4-position of the phenyl
where R4 and R5
i) are the same, either methyl or ethyl; or
ii) form an N-containing five-membered ring;
then R1 and R3 cannot simultaneously be H; and 
xe2x80x83in the 4-position of the phenyl
where R4 and R5, which are the same or different, are methyl or H; or
when R2 is xe2x80x94Oxe2x80x94CH2CH2xe2x80x94OH or xe2x80x94Oxe2x80x94CH2COOH in the 4-position of the phenyl, then R1 and R3, cannot simultaneously be H, or OH in the 4-position of the phenyl; and
if R1 is OH in the 4-position of the phenyl, R3 cannot be H.
According to another aspect the invention concerns a pharmaceutical composition comprising an amount effective to produce a tissue specific estrogenic and/or antiestrogenic effect of said novel selective estrogen receptor modulator compound, or a stereoisomer thereof, or a non-toxic pharmaceutically acceptable salt or ester thereof, and a pharmaceutically compatible acceptable carrier therefor.
According to an additional aspect the invention concerns a method of producing a tissue specific estrogenic and/or antiestrogenic effect in a subject in which such an effect is desired which comprises administering to said subject said novel selective estrogen receptor modulator compound, or a stereoisomer thereof, or a non-toxic pharmaceutically acceptable salt or ester thereof in an amount sufficient to produce the desired effect.
Additional embodiments and advantages of the invention will be set forth in part in the description as follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and not restrictive of the invention, as claimed.
This invention relates to the use of novel selective estrogen receptor modulators (SERMs) and their pharmaceutical preparations in men and women for the treatment of degenerative diseases and symptoms due to estrogen deficiency. Typically SERMs act as estrogens in bone and cardiovascular system while they are antiestrogenic in breast tissue. SERMs may have agonistic and antagonistic effects in other tissues also. Depending on their chemical structure and hormonal properties some compounds can be especially suited for elderly women for the prevention of osteoporosis whereas others (which are not feminizing estrogens) may also be used in men in the prevention of osteoporosis, cardiovascular diseases and Alzheimer""s disease. Some compounds are specifically suited for the treatment of climacteric symptoms in menopausal women. It is the common property of the described novel compounds that they are antiestrogenic in the mammary gland and inhibit the proliferation of breast cancer cells. They are also weak estrogens in the uterus and do not induce uterine cancers, the side effect of the well known SERM, tamoxifen.
The new SERMs of the present invention thus have tissue-specific estrogenic and/or antiestrogenic effects in vitro and in vivo and are useful in the prevention and treatment of osteoporosis, cardiovascular diseases and Alzheimer""s disease in men and women, as well as in the treatment of climacteric symptoms and breast cancer in women.
The compounds of formula (I) can be prepared by a process which comprises reaction of a compound of the formula 
where R7 is the same as R1 or R2 as defined before or is a protected such group, R3xe2x80x2 is R3 as defined before or a protected OH, R8 is benzyl or tetrahydropyranyl, with an organometallic compound of the formula 
where R9 is H , R1 or R2 as defined before or is a protected such group and M is xe2x80x94Mg-halogen or Li, to give a compound of the formula 
where R3xe2x80x2, R7, R8, and R9 are as defined above. R8 is tetrahydropyranyl when R7 or R9 is xe2x80x94Xxe2x80x94(CH2)nCH2xe2x80x94OR6 where X and n are as defined in (I). The compound (IV) is dehydrated by an appropriate acid catalyst preferable with acetic anhydride/acetyl chloride to give a triphenylethylene derivative of the formula 
where R8xe2x80x2 is H or benzyl, R7xe2x80x2 and R9xe2x80x2 are R1 and R2 or benzyl protected OH or benzyl protected xe2x80x94XCH2CH2OR6. The possible protecting tetrahydropyranyl groups in R3, R7, R8 and R9 are removed in this process to give radicals R3, R7xe2x80x2, R8xe2x80x2 and R9xe2x80x2.
The removal of the possible benzylic R8xe2x80x2 can be carried out by treatment with Zn and acetyl chloride in toluene to give the triphenylbutenol of the formula 
The hydroxy compound (VI) can be converted to a corresponding chloride by treatment with thionyl chloride or with triphenyl phosphine-carbon tetrachloride in organic solvent to give the compound of the formula 
The claimed compounds (I) are prepared from the compounds of the formula (VII) where R7xe2x80x2 and/or R9xe2x80x2 are benzyl protected xe2x80x94XCH2CH2OR6 by treatment with Zn and acetyl chloride in organic solvent or by catalytic hydrogenation.
Another process to prepare compounds of the formula (IV) is the hydroalumination reaction of a xe2x80x9cstyrenexe2x80x9d derivative of the formula 
where R10 is xe2x80x94CHO, xe2x80x94CH2OH, xe2x80x94COOH or a corresponding ester and R3 is as defined before with a benzophenone derivative of the formula 
Yet another process for the preparation of the compounds of the invention comprises O-alkylation of the compound of the formula (V) where R7xe2x80x2, and/or R9xe2x80x2 is OH with an alkyl halide derivative of the formula
R11-(CH2)m-halogenxe2x80x83xe2x80x83(X)
where m is an integer from 1 to 5 and R11 is halogen, 
where R6xe2x80x2 is R6 or protected R6, or xe2x80x94COOR to give a compound of the formula 
The compound of the formula (XI) where R11 is halogen is reacted with an amine of the formula 
to give a compound of the formula 
Yet another process for the preparation of the compounds of the formula (VII) comprises the McMurry reaction of an benzophenone derivative of the formula 
where R7xe2x80x2 and R9xe2x80x2 are as defined before, with an 3-chloropropiophenone derivative of the formula 
where R3 is as defined before.
The claimed compound of the formula (I) where R1 or R2 is 2,2-diethyl-2-hydroxyethoxy can be prepared by reaction of the compound of the formula (XI), where m is 1 and R11 is xe2x80x94COOR, with ethylmagnesium bromide.
The claimed compound of the formula (I) where R1 or R2 is 1-ethyl-2-hydroxyethoxy can be prepared by O-alkylation of the compound of the formula (V), where R7xe2x80x2 or R9xe2x80x2 is OH with ethyl xcex1-bromobutyrate and by reduction of the formed ester by lithium aluminum hydride.
Compounds of formula (I) contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers and other stereoisomeric forms. The present invention is also meant to encompass racemic mixtures, resolved forms and mixtures thereof as well as the individual enantiomers that may be separated according to methods known to those of ordinary skill in the art.
The invention disclosed herein is also meant to encompass non-toxic pharmaceutically acceptable salts and esters of compounds of formula (I) and their stereoisomers. The non-toxic pharmaceutically acceptable salts and esters can be prepared by methods well-known to those of ordinary skill in the art. The non-toxic pharmaceutically acceptable salts include, but are not limited to, chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, citrates, benzoates, salicylates, ascorbates and the like. The non-toxic pharmaceutically esters include, but are not limited to, methyl esters, ethyl esters, propyl esters, butyl esters and the like.
Evaluation of Estrogenic and Antiestrogenic Properties of Compounds in MCF-7 Cell Growth Experiments In Vitro
Estrogen-sensitive human breast cancer cells, MCF-7 (McGrath clone), were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 xcexcg/ml insulin and 10 xcexcg/ml gentamicin. The cells were grown as monolayer cultures in 75 cm2 plastic tissue culture flask (Nunc, Roskilde, Denmark) in 25 ml medium at 37xc2x0 C. in an atmosphere of 95% air, 5% CO2 and subcultured twice a week.
For experiments involving hormone or anti-hormone treatment, the cells in exponential growth phase were precultivated in the absence of estradiol for one day. Cells were plated at a density of 3.5xc3x97103 cells/well in 96-well microtiter plates (Nunclon, Roskilde, Denmark) and incubated for 24 hours at 37xc2x0 C., 95% air, 5% CO2, RPMI-1640 medium (L-glutamine and gentamicin as above) with 5% stripped fetal calf serum (stripped twice with dextran-coated charcoal to remove the steroids) and without phenol red. After the incubation period the medium was removed. The exposure to study drugs was started immediately by adding fresh medium with 5% stripped serum. Half of the cells were grown with estradiol, half without estradiol. Study compounds (dissolved in ethanol in 0.01 M concentration and diluted with the growth medium as appropriate) were added. The final concentrations of the compounds were 1, 10, and 100 nM, and 1 and 10 xcexcM. The cells were incubated for four days.
The amount of living cells was measured after 4 days by luminometer based on the amount of ATP and luciferase reaction as described by Kangas et al, 1984. This method allows evaluation of estrogenicity based on the ability of the compounds to stimulate the growth of the estrogen-dependent cells in the absence of estradiol. Estrogenicity was estimated by comparing the maximal growth stimulus (at any concentration) of study compound as per cent from growth stimulus by estradiol (100% stimulus). In the present studies antagonism was estimated at the concentration of 1 xcexcmol/l as per cent of theoretical full (100%) antagonism, which would mean complete inhibition of estradiol-stimulus. At high concentrations molecules may also show toxicity. Toxicity was estimated as the fraction of dead cells (i.e. 100% means that all cells have died during the exposure). The results are presented in Table 2.
Estimation of Estrogenicity and Antiestrogenicity In Vivo
The classical method to evaluate estrogenic and antiestrogenic effect is immature mouse or rat uterus (Terenius, 1971). The animals were exposed for 3 days to the compounds to be investigated at the age of 18 days. On the fourth day the animals were asphyxicated with CO2 and body weight and uterine weight was recorded. Estrogens increase the size and weight of the uterus (uterotropic effect) while antiestrogens inhibit this action. The compounds are therefore given alone and with estradiol in order to evaluate both agonistic and antagonistic effects. The results have been shown in Table 3 both as per cent of estrogen stimulation (100%), and as inhibition of estrogen action (full inhibition is 100%). The values are given at two dose levels, low i.e. 3-5 mg/kg and high i.e. 10-50 mg/kg. Estrogenic activity can be estimated also after a 4 weeks"" treatment of ovariectomized rats based on the uterine size. This assay was carried out in selected molecules as shown in Table 4.
Estimation of Effects to Cholesterol and Bone
Compounds were given p.o. to female rats for 4 or 5 weeks daily. At the end a blood sample was taken. Serum was separated by centrifugation and frozen until analyzed for total amount of cholesterol. Bone samples were taken from vertebra and tibia. Physical strength of the bones was studied as described by Peng et al, 1994. The assessments of the bone included:
Ash Weight of Tibial Epiphyses
Epiphyses of one tibia was carefully prepared and burned. Samples were burned to remove water and organic material. Ash weight relates to the mineral content of the bone. In addition, bone samples were taken to study the histomorphometry. In some cases the bone formation was studied by injecting tetracycline (50 mg/kg i.p. 10 days before autopsy) and calcein (20 mg/kg i.p. 3 days before autopsy). The method is based on permanent binding of tetracycline into growing bone and its detection by fluorescence (Peng et al, 1994).
Mechanical Testing of Bones
The mechanical testing of bones was carried out by materials testing machine, constructed in-house at Oulu University (Technical Services Department of the Medical Faculty). The testing machine is based on lever arm principle. One end of a steel lever is fixed. The pressing rod and the driving motor are connected to the lever arm with a moment ratio of 12.5 cm/50 cm=xc2xc. As a driving motor a linear actuator (SEY 10 Magnetic Elektromotoren AG, Switzerland) is used to obtain constant vertical movement (0.62 cm/s). The interchangeable compression head is mounted on the pressing rod for different tests transmitting compressive force to the specimen, and moving at a constant speed of 0.155 mm/s up to a maximal load capacity of 1200 N. The pressing rod is guided via an axial ball bearing to keep the movement vertical. Compressive force is measured by a temperature-compensated force sensor, which is attached to the stationary part of the compression stage. The measuring electronics include sensor calibration and adjustments.
Strength of Femoral Neck
The maximal load on the femoral neck was measured by the cantilever bending test. The supporter for the bone was a thick polymethyl methacrylate plate in which several holes of different sizes were drilled. On one side of each hole a groove was engraved for the third trochanter of the femur. The femur was cut exactly between the middle and lower third of the shaft. The bone was inserted perpendicularly and tightly into a suitable hole on the supporter. The lesser trochanter of each bone touched the surface of the plate. This procedure allowed rapid and stable fixation of the bone without using any additional embedding materials. The concave compressing head, 2.5 mm in diameter, was made of aluminum. The femoral head-neck complex was tested until failure by loading the head with a force parallel to the shaft.
Estimation of Antitumor Activity In Vivo
Antitumor activity was estimated by using DMBA (dimethylbenz[a]anthracene) model. One single peroral dose of DMBA (12 mg) initiates mammary gland carcinogenesis. New compounds were administered for 5 weeks when palpable tumors appeared. Size of the tumors and number of new tumors were carefully estimated once a week until termination. The model has been described in detail by Kangas et al, 1986. The growth of the tumors was measured once a week. All tumors were classified according to their growth properties to progressing, stable and regressing ones. Disappeared tumors were separately calculated. The tumors were considered to be progressing, if the tumor volume grew more than 8-fold during the 5 weeks dosing period, and regressing if the tumor volume decreased to one fourth or less from the volume in the beginning. If tumor volume changed less or remained unchanged, the tumors were considered to be stable.
Altogether 46 compounds were evaluated by the methods described above which are included in the list of example compounds numbered and listed in Table 1.
The structures of the example compounds are summarized as follows:
The estrogenic and antiestrogenic as well as cytotoxic effects of several compounds in vitro are presented in Table 2. It can be seen that the spectrum of hormonal activity of the compounds varies and thus gives the possibility to use the compounds in different clinical conditions.
Compounds with weak hormonal activity, which kill MCF-7 cells (human breast cancer cells) effectively at the highest investigated concentration (10 xcexcM) could be used preferably in the treatment of breast cancer. Such compounds are among others compounds No. 1, 3, 16, 19, 26, 27, 39 and 40 (Table 2). These compounds and several others are less effective estrogens and antiestrogens than the well known breast cancer drugs tamoxifen and toremifene (Table 3). Especially compound No. 19 is of interest, because it is a more effective anticancer drug in vivo in the DMBA-induced rat mammary tumor model even at very low doses than clinically used tamoxifen and toremifene (Table 6).
Compounds with weak estrogenic and no antiestrogenic action could be especially suitable for the prevention and treatment of osteoporosis and climacteric symptoms. Such compounds are (among others) compounds No. 3, 10, 11, 18, 19, 20, 25, 32, 36 and 44 (Tables 2, 3 and 4).
Compounds, which decrease cholesterol could be useful as cardiovascular drugs. For women some estrogenicity for such compounds can be allowed, but compounds which are not estrogens or are very weak estrogens and decrease cholesterol, could be used also in men for the prevention and treatment of cardiovascular diseases. Such compounds include (among others) compounds No. 3, 19, 20 (also for men) and 33 (for women) (Table 4). The same compounds are expected to be useful also in the treatment or prevention of Alzheimer""s disease. In the latter case the cytotoxic action of the compounds should be weak, like e.g. with compound No. 33 (Table 2). It should be noted that compound No. 19 does not show any estrogenic action on the weight of the prostate gland at doses which are active in DMBA-induced mammary tumor model (Tables 6 and 7). Therefore it could be of special benefit in men and could be of benefit in addition to the above mentioned conditions in the treatment of prostate cancer.
The hormonal profile of the compounds may be in some cases different in vitro and in vivo, e.g. compound No. 1 has no estrogenic action in vitro (Table 2), but is a weak estrogen in vivo (Table 3). Therefore the examples above should be understood as examples of the usefulness in different conditions. They should not be understood as limitations for their possible use in different clinical indications.
For the purpose of this invention, the novel SERMs, their stereoisomers or pharmaceutically acceptable salts thereof can be administered by various routes. The suitable administration forms include, for example, oral formulations, parenteral injections including intravenous, intramuscular, intradermal and subcutaneous injections; and transdermal or rectal formulations. Suitable oral formulations include e.g. conventional or slow-release tablets and gelatin capsules.
The required dosing of the novel SERMs will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, administration route and specific compounds being employed. Typically the daily dose for an adult person is 5-200 mg, preferably 20-100 mg. SERMs can be given as tablets or other formulations like gelatin capsules alone or mixed in any clinically acceptable non-active ingredients which are used in the pharmaceutical industry.